Results: Histologic review alone detected carcinoma with a sensitivity of 64% (39 of 61 cases) and 100% specificity. Quantitative real-time methylation-specific PCR for TIG1, APC, RARβ2, and GSTP1 detected carcinoma with a sensitivity of 70%, 79%, 89%, and 75%, respectively, with 100% specificity for all of the genes. Using this panel of methylation markers in combination with histology resulted in the detection of 59 of 61 (97%) cases of prostate with 100% specificity, a 33% improvement over histology alone.

Conclusion: The use of a panel of methylation markers as an adjunct to histologic review may substantially augment prostate cancer diagnosis from needle biopsies.

INTRODUCTION

Prostate adenocarcinoma is the most commonly diagnosed cancer in men in Western countries and the second leading cause of cancer related deaths in the United States. In 2002, an estimated 189,000 men were diagnosed with prostate cancer, and there were an estimated 30,200 deaths due to prostate cancer (1)
. Curative treatment entails radical prostatectomy or radical radiotherapy, and the best outcome is seen in patients with the earliest stage disease. In general, prostate cancer is diagnosed on needle biopsies taken to investigate a raised serum prostate-specific antigen or lower urinary tract symptoms. However, serum prostate-specific antigen can be increased in benign conditions, and biopsies may miss microscopic foci of cancer. Therefore, the introduction of additional diagnostic tests is needed to improve the sensitivity of prostate cancer diagnosis.

We have shown previously that the use of GSTP1 quantitative real-time methylation-specific PCR analysis can improve the sensitivity of prostate cancer diagnosis over histology alone (2)
. However, this marker is not present in all prostate cancer (between 90% and 95% of cancers). Therefore, we postulated that use of a panel of methylation markers might additionally improve sensitivity of prostate cancer diagnosis. We have reported previously that hypermethylation of TIG1(3)
, APC, RARβ2(4)
, and GSTP1(2)
are seen frequently in prostate adenocarcinoma. Therefore, we directly compared the sensitivity of quantitative real-time methylation-specific PCR for TIG1, APC, RARβ2, and GSTP1 methylation to standard histologic review of needle biopsies for detecting presence of prostate cancer.

MATERIALS AND METHODS

Patients and Sample Collection.

Fifty six patients undergoing prostatectomy for prostate adenocarcinoma and 16 patients undergoing cystoprostatectomy for bladder carcinoma at the Johns Hopkins Hospital between November 2001 and May 2002 (2)
were included in this study. Immediately after resection, sextant biopsies (apex, mid, and base from right and left sides) were taken from all 72 of the resected prostates and kept frozen at −80°C. The biopsies were sectioned to extract DNA with a 5-μm section taken every tenth slice and stained with hematoxylin and eosin for blinded histologic evaluation by an expert uropathologist (J. I. E.). All of the resected prostates were serially sectioned and examined histopathologically. These final pathology results were considered to be the gold standard for the presence of adenocarcinoma.

Bisulfite Treatment.

We extracted genomic DNA and carried out bisulfite modification of genomic DNA as described previously (5)
. Briefly, 2 μg of DNA in 20 μl of H2O containing 5 μg of salmon sperm DNA were denatured by incubation with 0.3 M NaOH at 50°C for 20 min. The DNA was then incubated at 70°C for 3 h in a 500-μl reaction mixture containing 2.5 M sodium metabisulfite and 0.125 M hydroquinone (pH 5.0). The treated DNA was purified with the Wizard DNA purification system according to the manufacturer’s instructions (Promega) and finally resuspended in 100 μl of H2O after ethanol precipitation.

Quantitative Real-Time Methylation Specific PCR.

DNA templates were amplified by fluorescence-based quantitative real-time methylation-specific PCR as described previously (5)
. Briefly, primers and probes were designed to amplify specifically bisulfite converted DNA at the 5′ end of TIG1, APC, RARβ2, GSTP1 and β-actin (used as the internal reference gene). The ratio of the gene of interest to β-actin (multiplied by 1000) for each sample was used as a measure for representing the relative level of methylated DNA for each gene of interest within each sample. The sequences of the primers and probe for TIG1 were 5′-TTTTTCGTCGCGGTTTGG-3′ (sense primer), 6-carboxyfluorescein-TCGGTTTTGCGTTGCGGAGGC-TAMRA (probe), and 5′-CGCTACCCGAACTTAATACTAAAATACG-3′ (antisense primer). The sequences for APC, RARβ2, GSTP1, and β-actin were described previously (6, 7)
. Amplifications were carried out in 384-well plates using a 7900 Sequence detector (Perkin-Elmer Applied Biosystems). All of the samples were run in triplicate, and each plate included multiple water blanks, a negative control, and serial dilutions of a positive control for constructing the calibration curve. Leukocyte DNA from a healthy individual was used as the negative control. The same lymphocyte DNA was methylated in vitro with excess SssI methyltransferase (New England Biolabs, Inc., Beverly, MA) to generate completely methylated DNA at all of the CpGs and used as the positive control.

Statistical Analysis.

We determined the medians and ranges of the methylation ratios for the samples. Associations between these values were tested by using the Mann-Whitney U test, and P values < 0.05 were considered to be significant.

RESULTS

We tested the same population of patients tested previously for quantitative real-time methylation-specific PCR of GSTP1(2)
. Final surgical pathology revealed five occult prostate adenocarcinomas in 5 of 16 cystoprostatectomy cases. Thus, our study included 61 true prostate cancer cases and 11 true negative (nontumor) cases. The pathological stages and grades of the 61 cases were shown in Table 1⇓
. A diagnosis of cancer was based on the requirement that only one of the six biopsies from each case needed to be called positive for the case to be positive.

Summary of clinicopathological features and promoter methylation status

First we performed a pilot study to determine specific analytical thresholds for TIG1, APC, and RARβ2 methylation. We checked the methylation status of 121 primary prostate cancer and 29 benign prostatic hyperplasia samples and established the threshold for each gene, which most efficiently distinguished cancer and benign prostatic hyperplasia samples (data not shown). Then we prospectively examined the biopsy samples using this threshold in blinded fashion. TIG1 quantitative real-time methylation-specific PCR detected prostate carcinoma with a sensitivity of 70% (43 of 61) and 100% specificity (11 of 11). This is a 6% improvement compared with histology alone (Table 1)⇓
. Representative results of quantitative real-time methylation-specific PCR for TIG1 were shown in Fig. 1⇓
. APC and RARβ2 quantitative real-time methylation-specific PCR showed 79% and 89% sensitivity with 100% specificity (Table 1)⇓
, representing 15% and 25% improvements respectively over blinded histologic examination. Fig. 2⇓
shows the highest methylation ratio in the sextant biopsies from each of the cases. The methylation ratio of all three of the genes (TIG1, APC, and RARβ2) revealed a significant difference between the cancer and nontumor groups (P < 0.0001).

Quantitative real-time methylation-specific PCR scatter plots of TIG1 (A), APC (B), and RARβ2 (C) in nontumor samples (N) and prostate adenocarcinoma (Ca). Measurements are expressed as a methylation ratio, defined as the ratio of the fluorescence intensity values for each gene to that of β-actin, multiplied by 1000. Quantitative real-time methylation-specific PCR revealed a significant difference in the ratio between the cancer and nontumor group in TIG1, APC, and RARβ2 (P < 0.0001).

As reported previously (2)
, blinded histologic assessment of the biopsies revealed 64% positivity (39 of 61) and 100% specificity (11 of 11). The combination of GSTP1 methylation and histology correctly diagnosed prostate cancer with 79% sensitivity, a 15% improvement. Individual analysis for each of the other three genes, TIG1, APC, and RARβ2, in combination with histology showed a much higher increase (18%, 21%, and 28%) in the sensitivity of prostate cancer diagnosis compared with histology alone (Table 2)⇓
.

To optimize the highest sensitivity of quantitative real-time methylation-specific PCR in diagnosis of prostate cancer, we checked a variety of combinations with these methylated genes. As shown in Table 2⇓
, the combination of TIG1 and RARβ2 showed the highest sensitivity of 95% (58 of 61) with 100% specificity, representing a 31% improvement compared with histology alone. Furthermore, using all four of the methylation markers, 97% of prostate cancers were detected, representing a 33% improvement in sensitivity compared with histology alone. With the combination, only 2 of 61 cancers remained undetected, and all of the benign samples were correctly identified as negative (Table 2)⇓
.

DISCUSSION

The preferred method for definitive diagnosis of prostate cancer is histologic analysis of sextant biopsies. Prostate needle biopsies provide not only histologic diagnosis but also additional information that is critical for management of prostate cancer patients (8)
. However, diagnosis of prostate cancer by biopsy can be difficult especially for small moderate-grade cancers (9)
. Needle biopsies only contain small samples of tissue and often include only a few malignant glands among many benign glands. Thus, it is not uncommon for many patients to be subjected to multiple biopsy examinations before a correct diagnosis is established. In this study, we tested the ability of a methylation panel to improve the sensitivity of standard histology for prostate cancer detection in needle biopsies. Using a combination of four genes, TIG1, APC, RARβ2, and GSTP1, all of which are frequently but not always methylated in prostate cancers, there was a large improvement in sensitivity to 95% with perfect specificity (Table 2)⇓
. A combination of all of the methylation markers demonstrated 97% sensitivity, yet 2 cases were still missed. These 2 missed cases harbored extremely small tumors, suggesting sampling error in the needle biopsies.

There are some limitations to this study. Firstly, histologic review of frozen sections is technically more difficult than paraffin sections and might partially explain the 22 missed cases by histologic analysis. However, we know that a significant number of prostate cancers is routinely missed at initial biopsy even using paraffin sections (8)
. Secondly, high specificity is important for any diagnostic test, because an established cancer diagnosis leads to major surgery and/or radical treatments with associated toxicities and side effects. Complete specificity was maintained using the methylation panel, but our number of negative cases was small, and this specificity needs to be more rigorously assessed in a larger number of negative cases. Thirdly, it is time to consider the cost of methylation marker assays in prostate cancer diagnosis. Quantitative real-time methylation-specific PCR assays are comparable with routine histologic assessment, but an increasing number of markers incorporated into clinical assays will increase the cost.

We have demonstrated the potential of adding gene methylation tests to routine histologic examination for the diagnosis of prostate cancer. Caution must be taken, because we must evaluate the newly tested methylated targets beyond GSTP1 in much larger numbers of normal control tissues and benign prostatic hyperplasia samples. Quantitative real-time methylation-specific PCR assays of key prostate cancer genes should be incorporated into larger diagnostic trials aimed at early disease detection. Validation of these assays in definitive studies could change the standard evaluation of sextant biopsies after routine prostate-specific antigen screening.

Footnotes

Grant support: National Cancer Institute Grant U01-CA84986.

Note: A senior editor of Clinical Cancer Research is a coauthor of this paper. In keeping with the AACR’s Editorial Policy, a member of the AACR’s Publications Committee had the article reviewed independently of the journal’s editorial process and made the decision whether to accept the article. Under a licensing agreement between Oncomethylome Sciences, SA and the Johns Hopkins University, Dr. Sidransky is entitled to a share of royalty received by the University on sales of products described in this article. Dr. Sidransky owns Oncomethylome Sciences, SA stock, which is subject to certain restrictions under University policy. Dr. Sidransky is a paid consultant to Oncomethylome Sciences, SA and is a paid member of the company’s Scientific Advisory Board. The term of this arrangement is being managed by the Johns Hopkins University in accordance with its conflict of interest policies.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.